1. Field of the Invention
This invention relates to radiodiagnostic reagents and peptides, and methods for producing labeled radiodiagnostic agents. Specifically, the invention relates to technetium99m (Tc-99m) labeled reagents, methods and kits for making such reagents, and methods for using such reagents. In particular, the invention relates to Tc-99m labeled somatostatin, derivatives of somatostatin, analogues of somatostatin or peptides that bind to the somatostatin receptor and contain at least 2 cysteine residues that form a disulfide or wherein the disulfide is reduced to the sulfhydryl form.
2. Description of the Prior Art
Somatostatin is a tetradecapeptide that is endogenously produced by the hypothalamus and pancreas in humans and other mammals. The peptide has the formula: ##STR1## [Single letter abbreviations for amino acids can be found in G. Zubay, Biochemistry (2d ed.), 1988, (MacMillan Publishing: New York), p.33]. This peptide exerts a wide variety of biological effects in vivo. It is known to act physiologically on the central nervous system, the hypothalamus, the pancreas, and the gastrointestinal tract. Somatostatin exerts it effects by binding to specific receptors expressed at the cell surface of cells comprising these organs. In addition, these high-affinity binding sites have been found to be abundantly expressed at the cell surface of most endocrine-active tumors arising from these tissues. Thus, the expression of high-affinity binding sites for somatostatin is a marker for these tumor cells, and specific binding with somatostatin can be exploited to locate and identify tumor cells in vivo.
One method that can readily be adapted to enable detection of tumor cells in vivo based on their expression of high affinity binding sites for somatostatin is radioimaging. Radionuclides which emit high energy gamma radiation can be readily detected by scintigraphy after injection into a human or an animal. A variety of radionuclides are known to be useful for radioimaging, including .sup.67 Ga, .sup.99m Tc (Tc-99m), .sup.111 In, .sup.123 I, .sup.125 I, .sup.169 Yb or .sup.186 Re. The sensitivity of imaging methods using radioactively-labeled peptides is much higher than other techniques known in the art, since the specific binding of the radioactive peptide concentrates the radioactive signal over the cells of interest, for example, tumor cells. This is particularly important for endocrine-active gastrointestinal tumors, which are usually small, slow-growing and difficult to detect by conventional methods.
Preparation of somatostatin analogues and uses for such analogues is known in the prior art.
Coy et al., U.S. Pat. No. 4,853,371 disclose synthetic octapeptide somatostatin analogues.
Coy and Murphy, U.S. Pat. No. 4,871,717 disclose synthetic heptapeptide somatostatin analogues.
Coy and Murphy, U.S. Pat. No. 4,485,101 disclose synthetic dodecapeptide somatostatin analogues.
Coy et al., U.S. Pat. No. 4,904,642 disclose synthetic octapeptide somatostatin analogues.
Taylor et al., European Patent Application No. WO 89/04666 disclose a method of treating cancer in an animal by administering at least a minimal dose of a hexapeptide analog of somatostatin.
Eck and Moreau, European Patent Application No. 90302760.5 disclose therapeutic octapeptide somatostatin analogues.
Methods for radiolabeling somatostatin analogues that have been modified so as to contain a tyrosine amino acid (Tyr or Y) are known in the prior art.
Albert et al., UK Patent Application 8927255.3 disclose radioimaging using somatostatin derivatives such as octreotide labeled with .sup.123 I.
Bakker et al., J. Nucl. Med. 31: 1501-1509 (1990) describe radioactive iodination of a somatostatin analog and its usefulness in detecting tumors in vivo.
Bakker et al., J. Nucl. Med. 32:1184-1189 (1991) teach the usefulness of radiolabeled somatostatin for radioimaging in vivo.
The use of chelating agents for radiolabeling polypeptides is known in the prior art.
Byrne et al., U.S. Pat. No. 4,434,151 describe novel homocysteine thiolactone bifunctional chelating agents for chelating radionuclides which can couple radionuclides to terminal amino-containing compounds capable of localizing in an organ or tissue which is desired to be imaged.
Fritzberg, U.S. Pat. No. 4,444,690 describes a series of technetium-chelating agents based on 2,3-bis(mercaptoacetamido) propanoate.
Gansow et al., U.S. Pat. No. 4,472,509 teach methods of manufacturing and purifying metal chelate-conjugated monoclonal antibodies.
Byrne et al., U.S. Pat. Nos. 4,571,430 and 4,575,556 describe novel homocysteine thiolactone bifunctional chelating agents for chelating radionuclides that can couple radionuclides to terminal amino-containing compounds capable of localizing in an organ or tissue which is desired to be imaged.
Nicolotti et al., U.S. Pat. No. 4,861,869 describe bifunctional coupling agents useful in forming conjugates with biologically molecules such as antibodies.
This reference describes compounds such as S-benzoylmercaptoacetylglycyl-glycylglycine.
European Patent Application 84109831.2 describes technetium chelating complexes of bisamido-bisthio-ligands and salts thereof, used primarily as renal function monitoring agents.
European Patent Application No. 86100360.6 describes dithio, diamino, or diamidocarboxylic acids or amine complexes useful for making technetium imaging agents.
European Patent Application 88104755.9 describes various S-protected mercaptoacetylglycylglycine chelating groups bound to large proteins such as antibodies.
Davison et at., Inorg. Chem. 20: 1629-1632 ( 1981 ) disclose a novel class of oxotechnetium chelate complexes.
Fritzberg et al., J. Nucl. Med. 23:592-598 (1982) disclose a technetium chelating agent based on N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate.
Byrne and Tolman, J. Nucl. Med. 24:P126 (1983) disclose a bifunctional thiolactone chelating agent for coupling Tc-99m to biological molecules.
Bryson et al., Inorg. Chem. 27:2154-2161 (1988) describe thiolate ligands for complexing with technetium.
Bryson et al., Inorg. Chem. 29:2948-2951 (1990) describe thiolate ligands for complexing with technetium.
Methods for radiolabeling somatostatin by covalently modifying the peptide to contain a metal chelating group has been disclosed in the prior art.
Albert et al., UK Patent Application 8927255.3 disclose radioimaging using somatostatin derivatives such as octreotide labeled with .sup.111 In via a chelating group bound to the amino-terminus.
Albert et al., European Patent Application No. WO 91/01144 disclose radioimaging using radiolabeled peptides related to growth factors, hormones, interferons and cytokines and comprised of a specific recognition peptide covalently linked to a radionuclide chelating group.
Kwekkeboom et al., J. Nucl. Med. 32:981 (1991) Abstract #305 relates to radiolabeling somatostatin analogues with .sup.111 In.
Albert et al., Abstract LM10, 12th American Peptide Symposium: 1991 describe uses for .sup.111 In-labeled diethylene-triaminopentaacetic acid-derivatized somatostatin analogues.
Methods for labeling peptides and polypeptides with Tc-99m have been disclosed in the prior art.
Dean, co-pending U.S. patent application Ser. No. 07/653,012 teaches reagents and methods for preparing peptides comprising a Tc-99m chelating group covalently linked to a specific binding peptide for radioimaging in vivo, and is hereby incorporated by reference.
Fritzberg, U.S. Pat. No. 4,444,690 describes a series of technetium-chelating agents based on 2,3-bis(mercaptoacetamido) propanoate.
Gansow et al., U.S. Pat. No. 4,472,509 teach methods of manufacturing and purifying Tc-99m chelate-conjugated monoclonal antibodies.
Reno and Bottino, European Patent Application 87300426.1 disclose radiolabeling antibodies with Tc-99m.
Pak et al., European Patent Application No. WO 88/07382 disclose a method for labeling antibodies with Tc-99m.
Rhodes, Sem. Nucl. Med. 4:281-293 (1974) teach the labeling of human serum albumin with technetium-99m.
Khaw et al., J. Nucl. Med. 23:1011-1019 (1982) disclose methods for labeling biologically active macromolecules with Tc-99m.
Byrne and Tolman, supra, disclose a bifunctional thiolactone chelating agent for coupling Tc-99m to biological molecules.
Cox et al., Abstract, 7th International Symposium on Radiopharmacology, p. 16, 1991, disclose the use of .sup.131 I- and .sup.111 In-labeled somatostatin analogues in radiolocalization of endocrine tumors in vivo by scintigraphy. Somatostatin labeled with technetium-99m under reducing conditions was used to scintigraphically localize colorectal carcinoma in rats following intravenous administration. Tc-99m labeled somatostatin was prepared by incubating the peptide with a solid phase electron donor and sodium pertechnetate at room temperature. Labeling efficiencies of 100% were obtained, with excess free technetium found to bind to the electron donor leaving only labeled complex in solution. Following intravenous administration in rats, rapid blood clearance was observed with accumulation in liver, kidneys and bladder; tumor uptake was found to achieve maximum levels at approximately 4 rain post-injection. Tumor-to-muscle uptake ratios were 5:1 which compared favorably with ratios of 3:1 reported for the .sup.131 I- and .sup.111 In-labeled analogues. The relationship of tumor label uptake to somatostatin binding was confirmed by a demonstration that somatostatin receptors blocked with suramin showed suppressed tumor uptake of the label.
The present invention provides peptides which are comprised of between 5 and 100 amino acid residues and at least 2 cysteine residues capable of forming a disulfide bond and that are labeled with Tc-99m. The preferred embodiments of the present invention are peptides that are somatostatin, derivatives of somatostatin, analogues of somatostatin or peptides that bind to the somatostatin receptor and contain at least 2 cysteine residues that form a disulfide or wherein the disulfide is reduced to the sulfhydryl form, and that are labeled with Tc-99m. Labeling with Tc-99m is an advantage of the present invention because the nuclear and radioactive properties of this isotope make it an ideal scintigraphic imaging agent. This isotope has a single photon energy of 140 keV and a radioactive half-life of about 6 hours, and is readily available from a .sup.99 Mo-.sup.99m Tc generator. Other radionuclides have effective half-lives which are much longer (for example, .sup.111 In, which has a half-life of 60-70 h) or are toxic (for example, .sup.125 I). Although Tc-99m is an ideal radiolabeling reagent, it has not been widely used in the an prior to the present invention [see, for example, Lamberts, J. Nucl. Meal. 32:1189-1191 (1991)].
Another advantage of the present invention is that the radioactively-labeled somatostatin, somatostatin derivatives, somatostatin analogues or peptides that bind to the somatostatin receptor and contain at least 2 cysteine residues that form a disulfide or wherein the disulfide is reduced to the sulfhydryl form provided by the invention have Tc-99m covalently linked to both of the cysteine-derived sulfur atoms capable of forming a disulfide bond of the peptide. Thus, the label can be directly linked to the peptide. The advantage over the prior art is that the invention avoids the necessity of providing somatostatin analogues which contain at least one tyrosine residue to enable radioactive iodine labeling. The invention also does not require the covalent linkage of heterologous chelating groups, described in the prior art. In addition, peptides according to the present invention can be prepared according to the methods of the invention from any available source of somatostatin, somatostatin derivative, somatostatin analog or peptide that binds to the somatostatin receptor and contains at least 2 cysteine residues that form a disulfide or wherein the disulfide is reduced to the sulfhydryl form, without the need to synthesize a derivative of a particular design or having specific metal ion chelating properties in addition to its biological specificity. The use of native somatostatin enabled by the invention also provides labeled peptides of well characterized binding specificity in vivo.
Thus the present invention provides Tc-99m radiolabeled peptides and radioimaging agents related to somatostatin and its derivatives or analogues, or indeed any peptide that binds to the somatostatin receptor and contains at least 2 cysteine residues that form a disulfide or wherein the disulfide is reduced to the sulfhydryl form, for use in imaging tumors and other tissues in vivo. The advantages of the invention include the use of Tc-99m as radionuclide, radiolabeling of native as well as modified somatostatin directly via the sulfhydryl groups of at least 2 cysteine residues of the peptide and the ability to utilize any available source of somatostatin, somatostatin derivative, somatostatin analog or peptide that binds to the somatostatin receptor and contains at least 2 cysteine residues that form a disulfide or wherein the disulfide is reduced to the sulfhydryl form, without requiring any particular modifications of the peptide.